Project description:Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC) or the lymphatic muscle (LMCs) cells themselves. Here we examined the cellular constituents of the mouse cLV wall and assessed whether any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a continuous if not contiguous network; spontaneous Ca2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreERT2 to target ICLC; PdgfrβCreERT2 to target pericyte like cells; PdgfrαCreERTM to target CD34+ adventitial cells and ICLC; and Myh11CreERT2 to target LMCs directly. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreERT2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreERT2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreERTM labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Of these cells, only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 under control of Myh11CreERT2, but not PdgfrαCreERTM or c-KitCreERT2, resulted in a propagated contraction upon photo-stimulation. Membrane potential recording in LMCs demonstrated that the diastolic depolarization was significantly correlated with contraction frequency. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.

Project description:While blood vessels have muscular walls that undergo tonic contractions to alter vascular resistance and, thus, control blood flow, lymphatics at the level of the collecting vessels and higher have muscular walls capable of rapid phasic contractions that generate lymph flow in addition to tonic contractions that regulate lymph flow resistance. While the ability of lymphatics to undergo rapid phasic contractions has been known for several centuries, the biological elements governing this phenomenon remain unknown. In an attempt to gain insight into the structural and regulatory elements that give lymphatic vessels their unique contractile capabilities, we utilized two-color microarray analysis to compare the thoracic duct of the rat to the vena cava of the same donor animal. Total cellular RNA was isolated immediately following vessel isolation and amplified in the presence of amino allyl dUTP. The resulting modified aRNA was conjugated to either Cy3 or Cy5 dye prior to hybridization to a rat 5.7K oligonucleotide array. Analysis and filtering of the data obtained from the microarray image yielded several contractile and regulatory genes with altered expression in the thoracic duct relative to the vena cava. Further evaluation of the data obtained in this study may aid in illustrating the unique properties of the lymphatic vessel and its muscular wall. Keywords: Thoracic duct, lymphatics, microarray Four unique thoracic duct/vena cava sample pairs were individually analyzed via two-color microarray analysis yielding 4 biological replicates. To minimize dye bias, a dye balance design was utilized in which the orientation of dye assignment was alternated between vessel pairs (i.e. two thoracic duct samples were labeled with Cy3 and two were labeled with Cy5). Prior to analysis, the data from 2 of the replicates was transformed to accomodate the dye balance such that all thoracic duct data is interpreted as Cy5 and all vena cava data is interpreted as Cy3.

Project description:The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.

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